The Refolding Kinetics Of Denatured Lysozyme Assisted By Fed-Batch Operation With Additives

This work studied refolding kinetics of denatured lysozyme by fed-batchoperation with dilution additives. The effect of the ratio of reduced glutathione (GSH)and oxidized glutathione(GSSG) on Artificial chaperone system (ACS) assistedlysozyme renaturation was investigated and the reaction condition was optimized.Then the effect of acidamide additives on lysozyme refolding was particular studied.A kinetic model was employed to analyze the renaturation process of denaturedlysozyme in the presence of additives. Based upon these, a novel renaturation methodusing fed-batch operation assisted by artificial chaperones was explored. Thecorrelative factors were also investigated. Through modifying the calculate method inthe batch period after the fed-batch operation, the refolding process kinetics model offed-batch operation was optimized. What's more, the experiment was employed toverify the corrected model. Followings are the result. At a proper concentration of GSH, the change of GSSGconcentration influenced the renaturation constant (KN) and aggregation rate constant(KA). At the proper ratio of GSSG by GSH, the ratio of KN by KA reached the maxvalue and the high refolding yield was obtained. Only change the GSH concentrationat fixed GSSG less influenced KN and KA. It was found that the refolding yield wasimproved in the presence of six aciamied additives, and there was an optimal additiveconcentration for each one. The refolding mechanism resembles that of guanidiniumchloride (GdmCl). That is, with increasing additives concentration, the KN decreased,but there existed special additives concentrations that minimized the KA, thus leadingto the highest refolding yield The high concentration of lysozyme (2mg/ml) completely refolded usingfed-batch operation assisted by artificial chaperones. Cetrimethyl ammomiumbromide (CTAB) is the important factor in the ACS system. There was an optimumconcentration of CTAB at fixed β-Cyclodextrin, which made the refolding to reachthe highest yield. What's more, there existed the co-operation effect of GdmCl andACS. The correted calculate method in the batch period after the fed-batch operationwas emplyed to simulate the refolding kinetics process at the low and high lysozymeconcentration, then the refolding process kinetics model was obtained. The model wasemployed to forecast the refolding yield at different lysozyme concentration underdifferent GdmCl concentration and fed-batch time. The experiment results verified themodel, which can accurately forecast the refolding kinetics of low and high lysozymeconcentration. So this model has the applicability.